This project has focussed on the study of protein oxidation in mammalian tissues during aging. The introduction of carbonyl groups as a marker for oxidative modification is a method extensively studied in this laboratory. It was demonstrated previously that the carbonyl content of proteins increases with age and in diseases causing premature aging. The liver, as the largest organ in the body, plays a central role in both metabolism and detoxification. The goal of this project is to identify highly oxidized proteins in the cell, understand their physiological function, and their course in aging. Using an immunological method for the detection of small amounts of oxidized proteins, we were able to identify an extensively modified protein, previously referred to as senescence marker protein (SMP-1) in rat liver. After purification by chromatographic methods, the protein was identified as carbonic anhydrase III (CAIII), the enzyme that catalyzes hydration of carbon dioxide. CAIII has been found by others to be extensively gluthathiolated protein, a modification thought to be important during oxidative stress. We have found that: 1. The level of carbonyl groups in CAIII, measured by 2,4- dinitrophenylhydrazine, is about 5-10 times that of the average protein. 2. The purified enzyme from old rats shows a clear increase in glutathione content, but no change in carbonic anhydrase activity. 3. The carbonyl content was unchanged during aging. 4. CAIII is inactivated in vitro when exposed to the metal-catalyzed inactivation system consisting in ascorbate/iron/oxygen.